Selection of synthetic proteins to modulate the human frataxin function

Frataxin is a kinetic activator of the mitochondrial supercomplex for iron-sulfur cluster assembly. Low frataxin expression or a decrease in its functionality results in Friedreich's Ataxia (FRDA). With the aim of creating new molecular tools to study this metabolic pathway, and ultimately, to explore new therapeutic strategies, we have investigated the possibility of obtaining small proteins exhibiting a high affinity for frataxin. In this study, we applied the ribosome display approach, using human frataxin as the target. We focused on Affi_224, one of the proteins that we were able to select after five rounds of selection. We have studied the interaction between both proteins and discussed some applications of this specific molecular tutor, concerning the modulation of the supercomplex activity. Affi_224 and frataxin showed a K_D value in the nanomolar range, as judged by surface plasmon resonance analysis. Most likely, it binds to the frataxin acidic ridge, as suggested by the analysis of chemical shift perturbations (nuclear magnetic resonance) and computational simulations. Affi_224 was able to increase Cys NFS1 desulfurase activation exerted by the FRDA frataxin variant G130V. Importantly, Affi_224 interacts with frataxin in a human cellular model. Our results suggest quaternary addition may be a new tool to modulate frataxin function in vivo. Nevertheless, more functional experiments under physiological conditions should be carried out to evaluate Affi_224 effectiveness in FRDA cell models.     

Molecular characterization of the proteinase-encoding gene, prb1, related to mycoparasitism by Trichoderma harzianum

The soil fungus Trichoderma harzianum is a mycoparasitic fungus known for its use as a biocontrol agent of phytopathogenic fungi. Among other factors, Trichoderma produces a series of antibiotics and fungal cell wall-degrading enzymes. These enzymes are believed to play an important role in mycoparasitism. Among the hydrolytic enzymes, we have identified a basic proteinase (Prb1) which is induced by either autoclaved mycelia, fungal cell wall preparation or chitin; however, the induction does not occur in the presence of glucose. The proteinase was purified and biochemically characterized as a serine proteinase of 31 kDa and pl 9.2. Based on the sequence of three internal peptides, synthetic oligonudeotide probes were designed. These probes allowed subsequent isolation of a cDNA and its corresponding genomic clone. The deduced amino acid sequence indicates that the proteinase is synthesized as a pre-proenzyme and allows its classification as a serine proteinase. Northen analysis shows that the induction of this enzyme is due to an increase in the corresponding mRNA level.     

Allosteric water and phosphate effects in Hoplosternum littorale hemoglobins.

This paper reports the results obtained using the osmotic stress method applied to the purified cathodic and anodic hemoglobins (Hbs) from the catfish Hoplosternum littorale, a species that displays facultative accessorial air oxygenation. We demonstrate that water potential affects the oxygen affinity of H. littorale Hbs in the presence of an inert solute (sucrose). Oxygen affinity increases when water activity increases, indicating that water molecules stabilize the high-affinity state of the Hb. This effect is the same as that observed in tetrameric vertebrate Hbs. We show that both anodic and cathodic Hbs show conformational substrates similar to other vertebrate Hbs. For both Hbs, addition of anionic effectors, especially chloride, strongly increases the number of water molecules bound, although anodic Hb did not exhibit sensitivity to saturating levels of ATP. Accordingly, for both Hbs, we propose that the deoxy conformations coexist in at least two anion-dependent allosteric states, T(o) and T(x), as occurs for human Hb. We found a single phosphate binding site for the cathodic Hb.     

The Murine Nucleolin Protein Is an Inducible DNA and ATP Binding Protein Which Is Readily Detected in Nuclear Extracts of Lipopolysaccharide-Treated Splenocytes

A 100-kDa DNA binding protein was found to be dramatically up-regulated upon the mitogenic stimulation of murine splenocytes with bacterial lipopolysaccharide (LPS). The induced DNA binding protein was also found to exhibit moderate binding specificity for the immunoglobulin isotype switch DNA repeats. Furthermore, the induction of the 100-kDa protein by LPS was found to be mediated by both an increase in the protein's stability and an increase in the synthesis of the protein. In vitro phosphorylation experiments revealed that the 100-kDa DNA binding protein was one of the most heavily phosphorylated proteins in both lymphoid and nonlymphoid nuclear extracts. Although this in vitro phosphorylation initially appeared to be mediated by a potent nuclear kinase activity, it was later determined that a significant part of the detected labeling was due to the direct binding of ATP by the 100-kDa protein. Antibodies raised to the 100-kDa DNA binding protein were used to isolate cDNA clones from a lymphocyte cDNA λgt11 expression library. Nucleotide sequence analysis revealed that the cloned cDNAs were identical to the mouse nucleolin gene. The β-galactosidase fusion proteins (encoded by exons 3-14 of nucleolin) and a more severely truncated 45-kDa protein (encoded by exons 5-14 of nucleolin) were both found to bind strongly to DNA and ATP. Furthermore, the strength of DNA binding was found to be highly dependent on the overall dG content of the DNA probes. Our experiments also revealed that apart from binding ATP and G-rich DNA, nucleolin directly bound GTP, dATP, and dGTP, but not dCTP, dTTP, or dUTP. Computer analysis revealed that the putative ATP binding domains appear to fall within two of the phylogenetically conserved RNA binding domains of nucleolin.     

Exudate-feeding byCallithrix jacchus penicillata in semideciduous woodland (Cerradão) in central Brazil

We report the exudate feeding behavior of two groups of marmosets (Callithrix jacchus penicillata) living permanently in Cerradão, a common woodland formation of Central Brazil. Cerradão is an open canopy formation and marmosets must occasionally descend to the ground in order to move from tree to tree. Even in atypical habitat, exudate eating is the predominant foraging activity. Marmosets are engaged in exudate collection over 70% of the total time spent feeding. They were observed gnawing on seven species of trees, and consumed exudates from four of these species. We compared the degree of utilization of the exudate sources, and examined a number of different characteristics of the exudates. Morphological adaptations that allow for the exploitation of the “exudate-eater niche” may be an important component of the adaptability ofCallithrix marmosets.     

Regulation of principal cell pH by Na/H exchange in rabbit cortical collecting tubule

Summary Changes in intracellular pH (pH _i ) were measured using the pH indicator, BCECF, in principal cells from split opened cortical collecting tubules (CCTs) derived from rabbits maintained on a normal diet. This monolayer preparation has the advantage of allowing us to visualize the morphological differences in the two major cell types in this nephron segment under transmitted light. The visual identification of the cell types was verified using emission measurements taken from single principal and intercalated cells in the opened tubule which had been exposed to fluorescein isothiocyanate (FITC)-labeled peanut lectin. We confirmed the existence of an amiloride-sensitive Na/H exchange process activated during intracellular acidosis in principal cells. In addition, the exchanger was active under basal conditions and over a wide range of pH _i . Because the exchanger was active under basal conditions we tested the hypothesis that changes in intracellular Na (Na _i ) would alter pH _i in a predictable way. Maneuvers designed to alter Na _i were without significant effects within a 10-min time frame. Specifically, addition of 100 m ouabain to increase Na _i or exposure of the tubules to 10^–5 m amiloride to decrease luminal Na entry and reduce Na _i did not have an effect on pH _i . In some experiments we did observe however, after a 30-min exposure to ouabain, a small decrease in pH _i . These results suggest that Na/H exchange is a major regulator of pH _i in principal cells. However, regulation of Na transport by changes in pH _i in principal cells of rabbit CCT via the activity of a Na/H exchanger do not seem to contribute to the feedback control of Na transport.This work was supported by U.S. Public Health Service grants DK27847 to L.G. Palmer and DK11489 to E.E. Windhager.     

DNA amplification fingerprinting of bacteria

Summary We have amplified short arbitrary stretches of total bacterial DNA to produce highly characteristic and complex DNA fingerprints. This DNA amplification fingerprinting (DAF) strategy involves enzymatic amplification of DNA directed by a single arbitrary oligonucleotide primer. Amplification produces a characteristic spectrum of products that is adequately resolved by polyacrylamide gel electrophoresis and visualized by silver staining. Although DAF is simple in concept, we found that amplification parameters must be within an optimal range for reproducibility. We establish a safe window for these parameters, which include magnesium, primer and enzyme concentration as well as cycle number. The refined procedure was used to distinguish between clinical isolates of Streptococcus uberis, Klebsiella pneumoniae, and Escherichia coli. The use of template DNA concentrations higher than 1 ng·l^–1 and high MgCl₂ levels was especially important for reproductibility when amplifying small bacterial genomes. We tested a truncated Thermus aquaticus DNA polymerase, the Stoffel fragment, and found it more tolerant of reaction conditions, more efficient in the amplification of short products, and able to produce more informative fingerprints when compared to the normal thermostable polymerase from which it was derived. Because DAF produces representative fingerprints quickly and reliably from bacteria regardless of prior genetic or biochemical knowledge, we anticipate the general use of this diagnostic tool for bacterial identification and taxonomy.Correspondence to: G. Caetano-Anollés     

Mechanisms of chromosome banding

Photo-oxidation of mitotic human chromosomes has been used in conjunction with anti-cytosine and anti-adenosine antibodies to produce R-banding. To elucidate the mechanism of this banding procedure we have examined the effect of photo-oxidation alone on chromosomes and nuclei. With short exposures to light in the presence of dilute methylene blue, C-band areas on chromosomes 1, 9, 16 and the terminal segment of the Y stain poorly. We call this phenomena reverse C-banding. After 18 h of exposure to light the chromosomes are swollen and show very little staining with quinacrine or Giemsa. Quantitative autoradiography shows that their DNA is almost completely extracted. Cytophotometric measurements also confirm that nuclear DNA is progressively extracted according to the length of exposure to light. When chromosomes are exposed to dilute methylene blue alone, without light, G-banded chromosomes result. We suggest the following explanation for these observations. In dilute methylene blue, C-band regions take up the greatest amount of dye and after short periods of photo-oxidation the DNA of these regions is preferentially destroyed resulting in reverse C-banding. Autoradiography in photo-oxidized chromosomes suggested that this preferential destruction of C-segments occurred in our experiments. With more prolonged exposure the DNA of the G-bands regions is preferentially destroyed and staining the remaining DNA with sensitive fluorescent labeled anti-C antibodies results in R-banding.     

On the fine structure of the electrocyte of Electrophorus electricus L.

Summary The general ultrastructure of the electrocyte, the basic unit of the electric organs of Electrophorus electricus, is analyzed. Presented herein are detailed observations of the syncytial surface, its fibrillar coat, invaginations of the plasma membrane and synaptic terminals. Using Thiéry's method glycogen granules were identified in the syncytial cytoplasm and inside the synaptic terminals, their size and structure being compatible with the muscular origin of the electric organs, to which the filamentous meshwork found in the cytoplasm may be related. Among the perinuclear-organelles, are dense bodies with crystalline patterns. The mitochondrial matrix contains dense granules, their size and structure varying according to the organ to which they belong and to the fixation method used.This work has been supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Conselho de Ensino para Graduados da UFRJ and Banco Nacional de Desenvolvimento Econômico, FUNTEC-241